The influenza B virus NB glycoprotein is abundantly expressed at the surface of virus-infected cells. NB spans the membrane once and has an 18 amino acid ectodomain, a 22 amino acid transmembrane domain, and a 60 amino acid cytoplasmic tail. The NB N-terminal ectodomain contains two asparagine residues that are modified by the addition of N-linked carbohydrate chains, which become further modified by the addition of polylactosaminoglycan. We have now shown that NB is also modified by addition of palmitic acid. To determine if NB is incorporated into virions, metabolic labeling, immunoblotting, and immunogold electron microscopy techniques were used. NB was identified in virions grown in MDCK cells or in embryonated chicken eggs in two forms: (a) NB modified by addition of polylactosaminoglycan (NB(pl)), and (b) a cleaved species (NB(c)) that has a smaller molecular weight than unglycosylated NB (NB12). Proteinase K digestion of purified virions converted NB(pl) to NB(c). Examination of virions purified by isopycnic centrifugation by electron microscopy and immunogold staining, using an affinity-purified antibody raised to a peptide derived from the NB cytoplasmic tail, showed staining for NB in influenza B virions. Quantification of the amount of NB in purified virions using two unrelated biochemical methods indicated there are on average approximately 15-100 molecules of NB per virion. Although the number of NB molecules incorporated on average into an influenza B virus particle is small, this finding is reminiscent of the number of molecules (14-68 monomers) found on average of the M2 integral membrane protein of influenza A virus.
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